The present invention relates to novel promoters, to novel chimeric genes containing them and to their use in plants for conferring to them an increased tolerance to herbicides. It also relates to the plant cells transformed by means of these genes and to the transformed plants regenerated from these cells as well as to the plants derived from crossbreedings using these transformed plants.
Glyphosate, sulfosate or fosametine are broad- spectrum systemic herbicides of the phosphonomethyl-glycine family. They act essentially as competitive inhibitors of 5-(enolpyruvyl)shikimate-3-phosphate synthase (EC 2.5.1.19) or EPSPS in relation to PEP (phosphoenolpyruvate). After their application to the plant, they are translocated inside the plant where they accumulate in the rapidly growing parts, in particular the caulinary and root apexes, causing the deterioration and even the destruction of sensitive plants.
Plastidial EPSPS, the main target of these products, is an enzyme of the aromatic amino acid biosynthesis pathway which is encoded by one or more nuclear genes and synthesised in the form of a cytoplasmic precursor and then imported into the plastids where it accumulates in its natural form.
The tolerance of plants to glyphosate and to products of the family is obtained by the stable introduction inside their genome of an EPSPS gene of plant or bacterial origin mutant or nonmutant with respect to the characteristics of the inhibition of the product of this gene by glyphosate. Given the mode of action of glyphosate, it is useful to be able to express the product of translation of this gene so as to permit its substantial accumulation in plastids.
It is known, for example from American U.S. Pat. No. 4,535,060, to confer to a plant a tolerance to a herbicide of the abovementioned type, in particular N-(phosphonomethyl)glycine or glyphosate, by introducing into the plant genome a gene encoding an EPSPS carrying at least one mutation making this enzyme more resistant to its competitive inhibitor (glyphosate), after localisation of the enzyme in the plastidial compartment. However, these techniques need to be improved in order to achieve greater reliability in the use of these plants under agronomic conditions.
In the present description, xe2x80x9cplantxe2x80x9d is understood as meaning any differentiated multicellular organism capable of photosynthesis and xe2x80x9cplant cellxe2x80x9d any cell derived from a plant and capable of forming undifferentiated tissues such as calluses or differentiated tissues such as embryos or plant sections, plants or seeds.
The subject of the present invention is the production of transformed plants having an increased tolerance to herbicides in general and particularly of the phosphonomethylglycine family by regenerating cells transformed by means of novel chimeric genes comprising a gene for tolerance of these herbicides. The invention also relates to novel chimeric genes as well as to transformed plants which are more tolerant due to better tolerance of the rapidly growing parts, as well as to plants derived from crossbreedings using these transformed plants. The subject of the invention is also novel promoters for constructing the above chimeric genes and comprising a DNA sequence capable of serving as promoter region in a chimeric gene which may be used for transforming plants, which comprises, in the direction of transcription, at least one promoter or a fragment thereof of a plant histone gene enabling the expression of the herbicide tolerance protein in the regions of accumulation of said herbicide.
More particularly, the subject of the invention is a chimeric gene for conferring to plants an increased tolerance to a herbicide whose target is EPSPS, comprising, in the direction of transcription, a promoter region, a transit peptide region, a sequence encoding a glyphosate tolerance enzyme and a polyadenylation signal region, wherein the promoter region consists of at least one fragment of a plant histone gene promoter enabling the preferential expression of a herbicide tolerance protein in the regions of glyphosate accumulation.
The histone gene is derived from a monocotyledonous plant such as for example wheat, maize or rice, or preferably from a dicotyledonous plant such as for example lucerne, sunflower, soya bean, colza or preferably Arabidopsis thaliana.CF Plant Mol. Biol Vol 8, 1987, p 179-191 Chaboute et al.
A histone gene of the H3 or preferably H4 type is preferably used, alone or under a multiplicated especially duplicated, form.
The promoter region of the chimeric gene according to the invention may in addition advantageously comprise at least one fragment of a promoter from a gene which is expressed naturally in plants, that is to say, for example, a promoter of viral origin such as the 35S RNA promoter of the cauliflower mosaic virus (CaMv35S), or of plant origin such as the small subunit of the ribulose 1,5-diphosphate carboxylase oxygenase (RuBisCO) gene from a crop such as for example maize or sunflower.
The transit peptide region comprises, in the direction of transcription, at least one transit peptide of a plant gene encoding a plastid-localised enzyme, a partial sequence of the N-terminal mature part of a plant gene encoding a plastid-localised enzyme and then a second transit peptide of a plant gene encoding a plastid-localised enzyme, preferably the RuBisCO gene.
The above transit peptides which can be used in the transit peptide region may be known per se and may be of plant origin, for example, derived from maize, sunflower, peas, tobacco or the like. The first and the second transit peptides may be identical, analogous or different. They may in addition each comprise one or more transit peptide units.
The partial sequence of the N-terminal mature part is derived from a plant gene encoding a plastid- localised enzyme, such as for example a maize, sunflower or pea gene or the like, it being possible for the original plant species to be identical, analogous or different from that from which the first and second transit peptides are derived respectively. Furthermore, the partial sequence of the mature part may comprise a varying number of amino acids, generally from 15 to 40, preferably from 18 to 33.
Construction of the entire transit peptide region may be known per se, in particular by fusion or any other suitable means. The role of this characteristic region is to enable the release of a mature protein with a maximum efficiency, preferably in native form.
The coding sequence for herbicide tolerance which may be used in the chimeric gene according to the invention encodes a mutant EPSPS having a degree of glyphosate tolerance. This sequence, obtained in particular by mutation of the EPSPS gene, may be of bacterial origin, for example derived from Salmonella typhymurium (and called in the text which follows xe2x80x9cAroA genexe2x80x9d), or of plant origin, for example from petunia or from tomatoes. This sequence may comprise one or more mutations, for example the Pro 101 to Ser mutation or alternatively the Gly 96 to Ala mutations. The untranslated polyadenylation signal region in 3xe2x80x2 of the chimeric gene according to the invention may be of any origin, for example bacterial, such as the nopaline synthase is gene, or of plant origin, such as the small subunit of the maize or sunflower RuBisCO.
The chimeric gene according to the invention may comprise, in addition to the above essential parts, an untranslated intermediate region (linker) between the promoter region and the coding sequence which may be of any origin, bacterial, viral or plant.